Represented by laser scanning confocal microscopy and two-photon microscopy, point scanning microscopic imaging technologies have the ability of three-dimensional sectioning imaging, thus have offered a wide range of applications in the researches of biomedical and materials science. Point scanning technologies get two-dimensional images at the focal plane of the objective lens by scanning the highly converged laser focus, and through axially scanning layer by layer to obtain the three-dimensional sectioned images of the sample. With the emergence of various new fluorescent molecular probes, the multicolor scanning microscopies allow the visualization of multiple protein interactions in living cells simultaneously. Besides, multicolor fluorescent labeling also provides improved imaging contrast and definition. High-end multicolor scanning microscopes developed so far are based on the multi-channel integration geometry. Multiple laser excitation sources and photomultiplier tube detectors for different color channels are employed, and the signals from each channel (red, green, and blue) are detected sequentially and combined into a single file. Laser scanning microscopies have axially sectioning capability and high spatial resolution, but scanning the entire three-dimensional samples point by point require a long time and the high power of laser may produce strong light damage and phototoxicity to living cells and tissues.
Differing from laser scanning imaging technologies, wide-field imaging can get all the two-dimensional information of the imaging plane by a single exposure using CCD or CMOS cameras. However, due to the certain depth of field of the objective lens, the image obtained by the CCD camera is actually the superimposing of the focal plane and the out-of-focus background. Due to the influence of the out-of-focus background, the image signal to noise ratio and the spatial resolution have been greatly restricted. Therefore, ordinary wide-field imaging cannot achieve three-dimensional sectioned images. Emerging in recent years, structured illumination microscopy (SIM) is a kind of wide-field optical microscopy, while has three-dimensional imaging capability. By projecting high spatial fringe pattern onto the sample, SIM can effectively separate the out-of-focus information and the in-focus information of the wide-field image by imaging processing algorithms. Scanning the sample along the axial direction of the objective lens by using the motorized sample stage, three-dimensional sectioned image can be obtained. Compared with laser scanning technologies, SIM has faster imaging speed and more compact configuration, light damage and phototoxity effect are also much slighter. Thus it's more suitable for in-vivo real-time imaging and research of biological tissues.
So far, most SIM systems use monochrome CCD or CMOS cameras to acquire images and cannot acquire the natural color information of the specimens. However, for a number of research areas (such as surface morphology measurements, materials science and other fields), it is very important to recover the color information of the sample. Although color sectioned images can be obtained via the use of confocal microscope under the principle of multicolor fluorescent labeling and multi-channel integration, the imaging speed is limited and the color is just the result of multicolor synthetic, not the real color.